The project objective is to identify and characterize genes involved in bacterial cell differentiation. Vegetative cells of the filamentous cyanobacterium Nostoc punctiforme (strain ATCC 29133) are capable of cellular differentiation into spore-like akinetes that function in perennation. N. punctiforme is an excellent model system in which to study cellular differentiation with respect to its recently sequenced genome, well developed genetic system, and availability of a newly developed DNA microarray. The first specific aim of this proposal is to identify genes differentially expressed during akinete differentiation using microarray analysis. This study will utilize a glucose-6-phosphate dehydrogenase mutant strain of N. punctiforme, designated UCD 466, that differentiates a large proportion of vegetative cells into akinetes following dark incubation in the presence of fructose. The wild-type strain is capable of heterotrophic growth under similar conditions and does not differentiate into akinetes. The DNA microarray technique will be performed with samples from various time points during akinete formation to identify regulated genes. The second specific aim is to construct and physiologically characterize mutants in selected differentially expressed genes by targeted gene replacement. Transcriptional reporter constructs will also be generated for selected genes and introduced into the wild-type and UCD 466 strains to determine temporal and spatial (cell-type specific) expression patterns. Promoter sequences from differentially expressed genes will be determined and the consensus sequence for common promoter elements used for future characterization of associated regulatory proteins. Functional characterization of differentially expressed gene products in akinetes will lead to a better understanding of how this specialized cell type can withstand harsh conditions. Regulatory mutants generated in this work will be used to identify regulons and regulatory cascades involved in akinete differentiation and allow identification of those that overlap differentiation processes in other cell types.